Countercurrent chromatography

INST R UMENTAT I0 N

Advisory Panel

I

Jonathah W. Amy Jack W. Frazer G. Phillip Hicks

Oonald R. Johnson Charles E . Klopfenstein Marvin Margoshes

Harry L. Pardue Ralph E . Thiers William F. Ulrich

Countercurrent Chromatography Development of support-free liquid-liquid partition techniques holds promise for preparative and analytical chromatographic separation of compounds without complications arising from solid supports YOlCHlRO IT0 and ROBERT L. BOWMAN Laboratory of Technical Development National Heart and Lung Institute Bethesda, Md. 20014

W H ?two ~

immiscible solvents containing solut,es are shaken in a separutory funnel and then separated, the solutes are partitioned between the two phases. The ratio of solute concentration in the upper phase t-o the lower is called the partit,ion coefficient. If the partition coefficients of two substances differ greatly, t.he only process needed for separation is a one-step operation called estraction. As the nature of the substances becomes more similar, the difference of their partition coefficient. decreases, hence requiring multistep extraction for separation. This t,echniquc, called the countercurrent distribution method ( I ) , can be performed with a Craig apparatus. Since the method is time-consuming and tends to dilute the sample, it is best suited for large preparative separation and is usually performed with no more than a few hundred glass partition tubes or ‘‘phtes.” On t,he other hand, high-efficiency microscale partition techniques, termed partition chromatography, involve a continuous partition process bet,ween the moving and the stat.ionary phases. The variety of methods developed elsewhere so far have employed solid supports (cellulose, silica, alumina, glass) to hold one phase st,:itionary. The granular and porous nature of the solid support provides an enormous surface area in relation to the liquid volume and divides the free space into thousands of plates. I n each plate, the partition process is theoretically completed, thus yielding an efficiency as high as thousands of theoretical plates. [--llthongh plate concept in chromatography is not cqitimlent t o that in the coiintercurrent distribution method, analogy may help to compare the efficicncy between these met,hocis (,?)I. Hon-cl-er, the affinitj- of the solid sup-

ports for the solute can add an undesirable ad,sorption effect evidenced by tailing of the elution curves of the solutes. When one deals with a minute amount of biological components, adsorption can result in a significant loss or denaturation of the sample in addition t o contamination by foreign materials elutcd from the support. General Features of Countercurrent Chromatography

K e have called our method countercurrent, chromatography to distinguish it from tlie countercurrent distribution method or conventional liquid partition chromntograpliy. Our system is similar to the c oun t ercu rren t dis:t ribu t ion method in that’ tlie t a o immiscible phascs pass t,lirough each other in a tnbular space. However, it involves a continuous nonequilibrium partition process comparable to chromatography. I t was dewloped t.o achieve a high-effi’ on ciency cliromatogrnphic separ a t ion both a preparative and analytical scale in the absence of solid supports. However, elimination of the solid support creates a number of problems as listed below :

(1) Hon- to keep the stationary phnse in the column as the moving phase is steadily eluted (21 Hoiv to divide the column space into numerous partition units and reduce laminar flow spreading of snmple bands (8) How to increase interfacial area ( 4 ) How to mis each phase to re-

duce mass transfer resistance. Several arrangements have been developed in our laboratory. each with a specific or potential advantage. I n each system, a tubular column is made to

form mnltiple traps to hold the st a t,ion’ a r y phase in a segmented pattern while a gravitational or centrifugal force maintains the two phnse states. The relative interface area is increased by dccrensing the tubular diameter and/ or increasing tlie number of the phase segments per unit, length of the column. In some cases, effective mixing is acconiplished by rotational or gyrational motion of the column, while the intcrface is held stnblc by gravity or centrifugal force. We will describe these arrangements and report their performancc to separate a test set of nine D S P (dinitropheiiyl) amino acids having p:irtition cocfficient,s ranging from >lo0 to 0.18, itsing a two-phase 3ystem composed of CHCIR, CH,COOH, and 0,lA‘ HCI ( 2 : 2 :1). Helix Countercurrent Chromatography (Helix CCC) (3, 4)

The method is so called becausc the two phases are formed in a helical tube, each phase occupying half the volume of each turn (Figure 1). The continiied injection of one phase causes the moving phase to percolate through the stationary ‘phase which is trapped by the effect of gravity. Solute locally introduced into t,he moving phase is exposed to each stationary segment, attaining a degree of equilibration dependent on the degree of mising that. results from the percolation, filming, and surface tension changes as the rolute is partitioned between the phases. To be effect.ive in the separation of materials with similar partition coefficients, a large number of partitioning exposures is required. This system has been tested with up to 17,000 turns of Teflon tithing of 0.2-mm i d . When small tubes are used, t,he cohesive

ANALYTICAL CHEMISTRY, VOL. 43, NO. 13, NOVEMBER 1971

69A

Instrumentation of the interfaces situated a t the top and the bottom of each coil unit (Figure IC) increases with the number of turns. Such interfaces not only contribute to the partition process but also prevent laminar flow spreading of sample bands. Hydrostatic pressure accumulated in the coil is found to be the limiting factor in this method and calculated from the equation:

P = RoZ(pl

Figure 1. Model of helix countercurrent Chromatography forces in the liquid exceed the gravitational forces tending t o separate the p h a e s . This necessitates the use of a centrifugal force field to maint,ain the phase separation and permit the percolation process to occur in fine tubes in a manner similar to that demoustrated in t,he large helix. In our prototype, t,he separat,ion tube is supported a t the periphery of a cylindrical centrifuge head and is fed from a coaxially rotating syringe. The plunger of the special high-pressure syringe is pushed through a thrust bearing by a multispeed syringe pusher. Pressures of u p to 20 atm were used in t,hese experiments, and we found it easier to push a rotating syringe than to make a high-pressure rotating seal. Effluent from the helix at approximately atmospheric pressure is conducted from the rotating system through a rotating s e d situated at the center of the t,hmst bearing. We have used two methods of coil preparation. I n the first,, the Teflon tubing is wound tightly on a flexible rod support which is subsequently coiled in a number of turns around the inside of the centrifuge head. Altemati,vely, the tubing is folded in two and twisted along its length t o provide a ropelike structure in which the individual strands have the appearance of a stretched helix of small diameter. Our experiments have shown that the efficiency increases with tube length, smaller bore, and slower flow. Also, with a given length of tubing, we can increase efficiency by increasing the number of coil units by decreasing the helix diameter. This may be reasonably explained by the fact that the number 70A

- p.)nd

(1)

where R denotes the distance between the center of the rotation and the axis of the helix; o, the angular velocity of rotation; n, the number of coil units; d, the helical diameter: and and ,", the density of lower and upper phases, respectively. Since the twisted configuration of the column described earlier gives a smaller helix diameter, d, t,han that of the ordinary coil, the twisted column has the potential for the highest efficiency by reducing the column pressure. Efficiency of over 8000 TP (theoretical plates) has been obtained on a twisted column of 17,000 turns prepared from 80 meters of 0.2-mm i.d. tubing. Seven D N P amino acids (each component 20 to 50 p g ) were eluted out within 30 hr at a flow rate of 125 pl/hr under a feed pressure of 16 atm. Droplet Countercurrent Chromatography (4, 5)

For preparative purposes, direct application of the model system (Figure 1) was found to he time-consuming, for the flow rate has to be low to prevent plug flow. When the helical configurat,ion was flattened by winding a tube onto a plate, introduction of a light phase wit,h low-wall surface affinit,y formed discrete droplets of the light

phase at the bottom of each coil unit. These droplets rose rapidly through the heavy phase at a regular interval with visible evidence of very active interfacial motion. Under ideal conditions, each droplet could become a plate if kept more or less discrete throughout the system. A system to exploit these ideas was developed by makmg long vertical columns of silanized glass tubing with fine capillary Teflon tubes t o interconnect the wider bore glass tubes. Discrete droplets, formed at the tip of the finer tube inserted at the bottom of the long glass tube, were made to follow one another with minimal space between and a diameter near that of the internal bore of the column (Figure 2). These droplets divide the column into discrete segments that prevent laminar flow along the length of the column as they mix locally near equilibrium. The fine Teflon tubing, inderconnecting the individual columns, preserves the integrity of the partitioning with minimal longitudinal diffusion and forms new droplets a t the bottom of the next column. The regularity of droplet size and close spacing, a droplet size nearly filling the bore, and thin return tubing, are important for best results. Our present column assembly consists of 300 glass tubes, 60-em long and 1.8-mm i.d., and has a total capacity of 540 ml. At a flow Tate of 16 ml/hr, several hundred milligrams of seven D N P amino acids can he eluted out within 80 hr a t an efficiency of 900 TP. Locular Countercurrent Chromatography (LCCC) (4) This method includes two varieties, rotation LCCC and gyration LCCC. Both systems employ a column pre-

Figure 2. Droplets in the column of droplet countercurrent chromatography Droplets formed a t the column junction can travel through the column length of 60 cm Without accident at a rate of about 2 cmlsec


Instrumentation

pared by placing multiple centrally perforated pnrt,it,ions across t8he diameter 'of a tubular column which divide the spnce into multiple cells called locides. I n each locule. the liquids form an interface while the solute partit,ioning is promoted by stirring of each phase induced by either rotation or gyration of the column. Rotation LCCC: Figure 3 illustrates the mechanism used for rotation LCCC. The column inclined at angle from the horizontal posit'ion is filled wit,h the loww phase, and the upper phase is fed t,hrough the first rot,atingseal connect,ion as the column is rota,ted around i t s o m auk. The upper phase then displaces the lower phase in each locule down to the level of the hole ~vliichleads to the next locule, the process being continued throughout the column. If the relationship betiyeen the iipper and the lower phases is reversed nt the beginning, t'he counterimilnrl!. proceeds. leaving the stationary iippcr phase in the c o l u n ~ n Conseqiimtly, solute introduccd into the column is subjected to :L multistep partition process, promoted by the rotation of the column, and finally cliitcd out through the second rotating-seal connection. Here, the gravity contributes to the phase separation, the column angle determines the phasc volume ratio in the locule, and the rotation of the column produces circular stirring of the liquids to promote partition. I n practice, we use a modified systrm in which mult,iple collimn units interconnected by fine Teflon tubings are mounted lengthwise on a rotating shaft, to produce a similar effect. Preliminary studies indicate that the efficiency increases with column lengt,h, and decreases with locule length and fast flow. The efficiency also increases with rotation speed up to 180 rpm or until t,he centrifugal force induced by rotation approaches the gravitat,ional force and the liquids are about to rot,at,e n-ith the column. The column angle determines bot,h volume ratio and interface area between two phases in the locule. The best result appears a t 30" where the volume rat8io of t,he two phases is nearly one, providing the maximal interface area. With a column consisting of 5000 locules, each 2.6 mm in diam and 3-mm long, and 1vit.h a total capacity of about, 100 ml, 1 ml of DKP amino acid sample is eluted out n-ithin 70 hr at an efficiency of 3000 TP or slightly over 50% partition efficienc>-/lociile. Gymtion LCCC; The second variety employs a vertical locular column which gyrates on a horizontal plane without rotational mot,ion (nonrota-

Figure 3. Mechanism of rotation locular countercurrent chromatography

tional gyration). The circular gyrational motion causes the interface to rotate inside the locule to produce necescary mixmg As the gyrating vertical column does not actually rotate, no rotating seals are necessary to permit flow in and out of the column during separation Figure 4A shows a diagram of the apparatus which provides a nonrotational gyration effect When a pair of large wheels rotates synchronously at mgular velocity w , a plate eccentri-

cally bridging these wheels conveys the motion to the column secured on the plate Radius T of the column gyration is equal to the distance on the wheel between the center and the eccentric connection I n our recent prototype, T has a fixed \ d u e of 2 5 ern and o is continuously adjustable up to 1500 rpm. The apparatus is installed in such a n a y that the axis of the gyration I.: held 1 ertical Figures 4B and C diow the effect of the nonrotational g:, ration on the

Figure 4. Mechanism of gyration locular countercurrent chromatography A. Diagram of t h e apparatus B. Gyration effect on liquid interface i n locule on cross section of successive positions of the s a m e locule C. Gyration effect o n liquid interface i n locule on vertical section


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Instrumentation

Figure 5 ( A thru E). Principle of countercurrent chromatography with flowthrough coil planet centrifuge The figures schematically illustrate the general principle and the motion o f the two phases in a coiled tube undergoing planetary motion

liquids and the int,erface present in an individual locule, The cross section (Figure 4B) through a middle portion of the locule shows successive positions of one locule as it is gyrated about a cent8ral point.. The upper and lower phases are seen separated b y centrifugal force forming a circular interface perpendicular to the action of the force. With the steadily changing direction of the centrifugal force, both liquids and interface rot,ate with respect to the “x” fixed to the column wall. Thus, friction between t,he liquids and the internal surface of the locule induce stirring in each phase to accelerate the partition process. On t h e vertical section (Figure 4C) through the center of the locule, the liquids form a parabolic interface perpendicular t,o the vect,or given by summation of the centrifugal and gravitat,ional acceleration. The angle Q: here again determines both volume ratio and interfacial area bet,vveen the two phases in the locule. Since t h e moving phase usually covers t,he entire area of t,he exit hole in the locule, the maximum interface area is given by moving either the upper phase upward or the lower phase downward t,hrough the column. Preliminary experiment,s have shown that,, in general, the efficiency rises with column length, number of locules, slower flow, and faster gyration. C a r r y o v e r of t h e stationary phase, which tends t80increase with vibration of the apparat.us a t a high-gyration speed, is found to be the limiting factor in this met.hod. Two devices have been successful in eliminating t,his complicat.ion. T h e use of a column with threaded partition holes prevents the carry-over, if the direction of gyration is chosen to screw t,he carried-over droplets back to the original locule,

Also, we have found t.hat the surface nature of the partition hole great,ly relates to this phenomenon. When the exit hole of the locule is made of a material wett,able by the moving phase, the bore becomes smaller, there is less tendency of carry-over, and higher efficiency is expected. To make the locular column more convenient for the choice of the moving phase (eit,her aqueous or nonaqueous phase may be chosen), we embedded small glass beads (1.2-mm o.d., 0.5-mm i.d., 0.7 mm long) partially into each Teflon partition hole to make one side of the hole wettable to the aqueous phase and the other wettable to the organic phase. Use of such partitions completely eliminates carryover of the stationary phase at 800 rpm and yields a high partition efficiency from 100% to 40% in each locule. Countercurrent Chromatography with Flow-Through Coil Planet Centrifuge (6)

This method stems from a partition technique utilizing a coil planet centrifuge ( 7 , 8) which induces a planetary motion to a helical tube in a manner such that the rotat’ion of the helical tube is extremely slow in comparison with the revolution. I n such a system, however, flow becomes difficult’ because of the need for t,he rotating seals; therefore, the separat.ion is usually performed within a closed helical t,ube. Thus, problems of sample introduction and fractionation limit the practical use of the method. On the ot’her hand, the present method introduces a continuous flow-through system t o the coil planet, centrifuge without rotating seals. Figure 5 A , B illust,rates the general principle of the method. The separation column is a helix, as indicated in Figure 5A. Flexible feed and return tubes are supported by the moving

disc at, the top of t.he helix and t,he stationary disc fixed to the center of the upper frame of the centrifuge. The entire helix and the moving disc revolve around t’he axis of the centrifuge, but they are not permitted t,o rotate with respect to the stationary disc. The fixed orientation of the helix is maintained by coupling a pulley on the helix holder through a toothed belt to a stat.ionary pulley of equal diameter on the axis of the centrifuge drive. This coupling causes a counter rotation (-W) of the helix to cancel out’ the rotation (0)of the helix induced by its revolution. The feed and return tubes do not twist because the moving disc does not rotate with respect to the stationary disc a.s indicated in Figure 5B by the position of the “x” marks shown in successive positions of the relation between the stationary and moving discs. Because the helical column maintains a fixed orientation while it revolves, the ra,dially directed centrifugal force rotates with respect to the column as in the gyrating system described earlier. Let, us consider the motion of t,he two immiscible phases, t,he upper and lower phases, confined in such a tube without the external introduct,ion of flow (Figure E, D, and E ) . For the convenience of illustration, the direction of the centrifugal force which actually rotates is fixed a t the bottom and, instead, the rotation of the coil units is substituted as indicated by the curved arrows. Figure 5C shows the motion of a small amount of the lower (heavier) phase in the upper phase. If t.he relative centrifugal force, Rw2, is strong enough t’o keep the lower phase near the bottom of the coil unit ( a ) a t all times, the latter will move steadily toward one end of the coil, the head (the other


73A

Instrumentation

end is called the tail), a t angular velocity W . I n the present system, the centrifugal force usually fails to fix the lower phase which subsequent,ly appears at any portion of t,he coil unit. When the lower phase appears a t the left half of the coil unit ( b ) , it tends to move toward the head, while, at the right) half (e), it moves toward the tail. Because the lower phase t,ends to spend more time in the left half, these t'mo motions do not cancel out, and the lower phase moves toward the head with an oscillatory motion a t a mean angular velocit,y smaller than W . Figure 5 0 illustrates the motion of a, small amount of the upper phase in t,he lower phase. With a strong centrifugal force, the upper phase could be fixed near t,he t'op of the coil unit ( a ) , constantly moving toward the head of the coil. When the centrifugal force fails t o fix the upper phase, the latter moves toward the head at the right half ( b ) and toward the t,ail a t the left half (c) of the coil unit. However, these motions again do not cancel out, and the upper phase also moves toward the head. When a similar volume of t,he two phases is introduced into the tube, the motion of the phases becomes quite complex but, finally reaches an equilibrium state, illustrated in Figure 5E. At, the equilibrium, the multiple segments of the t>wophases are alt'ernately arranged from the head to the tail side, and any excess of eit,her phase remains at' the tail. (Wit,h a. relatively largebore tubing, a st,rong centrifugal force field may produce multiple droplets of either phase in t.he other. The picture of t,his hydrodynamic phase equilibrium may be modified by various factors such as viscosity, interfacial t,ension, wall surface affinity, densit.y difference of the two phases, etc., and for some sol-

-P

-

A

0

stationary tube and the other at the bottom of the coil holder, are connected by a t,oot,hed belt, revolution introduces the desired planetary motion to the coil holder-Le., one rotation per revo1ut)ion in the opposite direction. The column is made of Teflon tubing either by winding the tube onto the coil holder or by arranging multiple column units interconnected in a series (tailhead connection) or several separate parallel columns around t'he holder. Both feed and return tubes are passed through the center hole at the top of t'he holder and then supported a t the level of 25 cm above t,he cent'er of the apparatus. These tubes, protected with a piece of silicone rubber tube a t the hole, have not failed or stretched appreciably for many runs. Figure 6A shows separation for nine DNP amino acids on an analytical column of 0.30-mm bore Teflon tubing, 100 meters long with a helix diameter of 5 mm and a total capacity of about 8 ml. Sample size is 10 pl containing each component a t about, 1% where solubilit,y permits. The upper phase is fed with a metering pump a t a rate of 2.4 ml/hr while the apparatus is spun at 550 rpm with a radius of 30.7 cm, the equilibrium feed pressure being approximately 200 psi. The efficiency ranges between 10,000 and 3000 TP, showing a tendency to decrease with increased retention time. Figure 6B shows a chromatogram obtained on a preparative column prepared from 1.4mm bore tubing, 100 meters long with a helix diameter of 1 cm and wit,h a tot'al capacity of about 140 ml. At 520 rpm with a n 8.6-cm radius of revolution and at a flow rate of 60 ml/hr, a sample size of 2 ml can be eluted out within 7 h r a t an efficiency ranging bet'ween 2000 and 500 TP.

vent systems, the ideal condibion may be obtained by using an even greater radius of revolut.ion than that used in the present test system.) Consequently, this phenomenon determines the proper direction of elution. When the tube is filled with either phase and the other is introduced from the head, the equilibrium process is quickly established from the head through the tail as elution proceeds and a given volume of the shtionary phase is held within the equilibrated coil (Figure 5 A ) . On the other hand, introduction of the flow through the tail results in a steady carry-over of the stat,ionary phase until the moving phase fills the entire column. Thus, the moving phase should be fed from the head end, determined by both handedness of the coil and direction of the planetary motion. Consequently, a sample solution int.roduced into the tube is subjected to a partition process between the oscillating alternate segments of t,he two phases and finally elut,ed out through the tail end of the coil. To form the two phases into multiple segment.s in a small-bore tubing without plug flow, an adequate centrifugal force is provided by the relatively long radius of revolution. We have const,ructed a test system with radii adjust,ableto 30.7, 20.2, 13.5, and 8.6 ern by modifying a conventional centrifuge. The motor shaft, is extended through the top-end bearing of a stationary tube (60 ern long) mounted to the motor housing and then connected t,o a rotating tube that fits freely over the stationary tube with another bearing at the bottom. il pair of arms fixed to the top and bott.om of the rotating tube holds the rotating rods or coil holders wit,h bearings. When a pair of toothed pulleys of the same size, one fixed to the bottom of the

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80

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TIME (hours)

Figure 6. Results of DNP amino acid separation.

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TIME (hours)

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70

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Recording was originally made by LKB uv monitor (Uvicord II) a t 280 nm. Identification of the peaks and their partition coefficients are given on the chart A. Analytical chromatogram B. Preparative chromatogram


Instrumentation Conclusions

The various syst,ems developed for countercurrent chromatography show high reproducibility and meet prepa.rative and analyt,ical needs. The advantages of countercurrent chromatography over conventional liquid part’ition techniques are briefly discussed below. Compared with t.he count’ercurrent distribution method (CCD), countercurrent chromatography practically yields more theoretical plates and reduces the degree of sample dilution occurring during separation. The t’ime required for separation is also lessened. The values of countercurrent chromat,ography analogous to one transfer time in CCD, obtained by dividing the retention time of the solvent front by the yielded t,heoretical plate number, range from 40 see (droplet CCC) t,o 1 see (flow-t,hrough coil planet centrifuge technique), whereas in CCD, one transfer requires several minutes. Thus, the present method is suit,able for separation of samples on the order of 10 mg, which is just enough to complete most biochemical in\-estigations. On the other hand, countercurrent chromatography eliminates complications arising from the use of solid supports in conventional partition chromatography, hence tailing of t,he solute peaks, sample loss, denaturation, and contamination are minimized. Also, this met.hod of countercurrent chromatography enables us to predict 1oca.tions of t,he eluted solute peaks once their partition coefficient,s are known. Thus, it niny be extremely useful for purification of a minute amount of biological material from a crude mixture. Countercurrent chromatography, as introduced here, is a new development. K h e n the instruments and techniques are further refined, the met,hod will be even more useful. Even now, t,he technique offers many unique advantages over other separation methods, References

(1) L. C. Craig, “Comprehensive Biochemistry,” Vol 4, Elsevier, Amsterdam, London, and New York, N.Y., 1962. (2) A. I. hI. Keulemans, “Gas Chromatography.” Chapt. 4, Reinhold, New York, N.Y., 1957, pp 96-129. (3) Y . Ito and R . L. Bowman, Science, 167,281 (1970). (4) Y. Ito and R . L. Bowman, J . Chromcctogr. sci.,8, 315 (1970). ( 5 ) T. Tanimura, J. J. Pisano, Y. Ito, and R . L. Bowman, Science, 169, 54 (1970). (6) Y. Ito and R . L. Bowman, Science, 173,420 (1971). (7) Y. Ito. M. -4.Weinstein, I. Aoki, R. Harada, E . Kimura, and K. Nunogaki, A\rfllure, 212, 985 (1966). ( 8 ) Y. Ito, I. Aoki, E . Kimura, K. Nunogaki, and Y. Nunogaki, ANAL. CHEM., 41, 1579 (1969).

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75A

Countercurrent chromatography

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